Cloning of the human alpha 1d-adrenergic receptor and inducible expression of three human subtypes in SK-N-MC cells

We have cloned the human Alpha 1d-adrenergic receptor (AR) and compared the pharmacological properties of the three Recombinant human Alpha 1-AR subtypes in SK-N-MC cells. SK-N-MC cells natively express a mixture of Alpha 1-AR subtypes, and the use of an inducible expression system allowed us to directly compare the Recombinant and native subtypes without concern for cell-specific processing or microenvironment. The human Alpha 1d-AR was expressed from a cDNA/gene fusion construct cloned from human SK-N-MC cell cDNA and human genomic libraries. This receptor is deduced to contain 572 Amino acids with 98% identity to the rat Alpha 1d-AR in the transmembrane domains and, when expressed in human embryonic kidney 293 cells, has Alpha 1-AR binding properties similar to those of the rat Alpha 1d-AR. Norepinephrine increased inositol phosphate formation and mobilized intracellular Ca2+ in transfected 293 cells. Reverse transcription-polymerase chain reaction analysis of the three cloned human subtypes (Alpha 1a, Alpha 1b, and Alpha 1d) in mRNA from SK-N-MC cells, which natively express Alpha 1A- and Alpha 1B-like pharmacology, showed abundant Alpha 1a and Alpha 1d but fewer Alpha 1b transcripts. The three human clones were expressed in SK-N-MC cells using isopropyl-beta-D-thiogalactoside-inducible vectors. Upon induction, Alpha 1-AR density was increased with the Recombinant subtype comprising 67-80% of total Alpha 1-ARs. Inhibition curves for (+)-niguldipine and 5-methylurapidil fit best to a two-site model in uninduced cells, indicating significant receptor heterogeneity. Isopropyl-beta-D-thiogalactoside induction altered the potencies of both compounds, causing most inhibition curves to fit best to a one-site model. (+)-Niguldipine was 100-fold more potent at the Alpha 1a-AR than at Alpha 1b- or Alpha 1d-ARs, whereas 5-methylurapidil had similar potencies at Alpha 1a- and Alpha 1d-ARs and about 10-fold lower affinity at the Alpha 1b-AR. We conclude that the complex Alpha 1A- and Alpha 1B-like pharmacology observed in native SK-N-MC cells is due to expression of all three subtypes in different proportions, independently of cell-specific processing or environmental factors, and that the Alpha 1a-AR cDNA encodes the pharmacologically defined Alpha 1A subtype.